U.S. patent application number 11/573319 was filed with the patent office on 2008-08-28 for assay device & method.
This patent application is currently assigned to Inverness Medical Switzerland GmbH. Invention is credited to David Edward Williams, Krzysztof Wojciech Zak.
Application Number | 20080206849 11/573319 |
Document ID | / |
Family ID | 32982678 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080206849 |
Kind Code |
A1 |
Zak; Krzysztof Wojciech ; et
al. |
August 28, 2008 |
Assay Device & Method
Abstract
There is disclosed a lateral flow assay device and method of
producing the same. The device, which is for identifying
carbohydrate antigens in a biological sample comprises a substrate
having, a) a sample receiving zone, b) an extraction zone for
receiving the sample from said sample receiving zone and which
extraction zone comprises immobilised or otherwise absorbed therein
at least one or more reactants and reagents which when combined
react to form an extraction reagent for a desired antigen in said
sample, said sample to be added to said sample receiving zone
optionally comprising the remaining reactant required to form said
extraction reagent if not present in said extraction zone, c)
optionally a neutralising agent capable of bringing the pH of the
resulting sample to within the operational pH range of the assay
and, d) a detection zone for a labelled specific binding or capture
reagent for said antigen to be detected.
Inventors: |
Zak; Krzysztof Wojciech;
(Bedford, GB) ; Williams; David Edward; (St.
Heliers, NZ) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP (w/ISA)
155 SEAPORT BLVD.
BOSTON
MA
02210-2600
US
|
Assignee: |
Inverness Medical Switzerland
GmbH
Zug
CH
|
Family ID: |
32982678 |
Appl. No.: |
11/573319 |
Filed: |
July 27, 2005 |
PCT Filed: |
July 27, 2005 |
PCT NO: |
PCT/GB2005/002927 |
371 Date: |
April 24, 2008 |
Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
G01N 2469/10 20130101;
G01N 2400/00 20130101; G01N 33/558 20130101; G01N 33/56944
20130101 |
Class at
Publication: |
435/287.2 |
International
Class: |
C12M 1/00 20060101
C12M001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 6, 2004 |
GB |
0417601.2 |
Claims
1. A lateral flow assay device for identifying carbohydrate
antigens in a biological sample, said device comprising a substrate
having a) a sample receiving zone, b) an extraction zone for
receiving the sample from said sample receiving zone and which
extraction zone comprises immobilised or otherwise absorbed therein
at least one or more reactants and reagents which when combined
react to form an extraction reagent for a desired antigen in said
sample, said sample to be added to said sample receiving zone
optionally comprising the remaining reactant required to form said
extraction reagent if not present in said extraction zone, c)
optionally a neutralising agent capable of bringing the pH of the
resulting sample to within the operational pH range of the assay
and, d) a detection zone for a labelled specific binding or capture
reagent for said antigen to be detected.
2. A device according to claim 1 wherein said extraction zone
comprises both said precursor reactants that when combined react to
form said extraction reagent.
3. A device according to claims 1 or 2, wherein said antigen to be
detected is a Streptococcal antigen.
4. A device according to claim 3, wherein said Streptococcal
antigen is Streptococcal A antigen.
5. A device according to any preceding claim wherein said
extraction reagent is a nitrous acid, each of said precursor
reactants being an acid and a nitrite and which when combined form
the nitrous acid.
6. A device according to claim 5, wherein said acid is a sulfonic
acid in the form of a resin and said nitrite is sodium nitrite.
7. A device according to any preceding claim wherein said substrate
comprises a lateral flow porous carrier.
8. A device according to any of claims 1 to 6 wherein said
substrate comprises microfluidic channels the dimensions of which
are such as to allow a liquid sample move there through by means of
capillary action.
9. A device according to any preceding claims wherein said labelled
specific binding reagent is immobilised at or upstream of said
detection zone.
10. A device according to any of claims 1 to 9, wherein said
labelled specific binding reagent is added to said substrate
together with or after addition of said sample.
11. A device according to any of claims 1 to 9 wherein said
substrate comprises a mobilisable labelled specific capture reagent
and provided at a position downstream from the optional
neutralising agent.
12. A device according to claim 11, wherein the mobilisable capture
reagent is provided on and/or in a separate porous carrier arranged
so as to be in fluidic contact with and upstream from the porous
carrier.
13. A device according to claim 12, wherein said separate porous
carrier is macroporous and may be of a different material to that
of the porous carrier.
14. A device according to any preceding claim wherein said labelled
specific binding reagent is labelled antibody.
15. A device according to any preceding claim where said label is
an enzyme label or a specific dye.
16. A device according to any preceding claim, wherein said sample
receiving zone comprises a sample receiving wick made of any
bibulous, porous or fibrous material capable of absorbing liquid
sample.
17. A device according to any preceding claim, wherein the porous
carrier is a nitrocellulose carrier.
18. A device according to claim 17, wherein said porous carrier is
nitrocellulose and the macroporous carrier is selected from any of
glass fibre of a synthetic polymeric material such as
polypropylene.
19. A device according to any preceding claim, wherein said
substrate comprises an absorbent sink at the distal end
thereof.
20. A device according to any preceding claim further comprising a
control zone at or downstream from the detection zone and which
comprises a further immobilised capture reagent that is able to
capture the labelled capture reagent.
21. A kit for identifying carbohydrate antigens in a biological
sample, which kit comprises a device according to any preceding
claim and means for contacting said sample with said sample
receiving area.
22. A method of producing an assay device according to any of
claims 1 to 20, which method comprises immobilising in a lateral
flow assay device having a sample receiving area, one or more
reagents in an extraction zone of said device and which reagents
when combined form an extraction reagent for any carbohydrate
antigen in a sample added to said sample receiving zone, providing
a neutralisation buffer downstream of said extraction zone to
neutralise the pH of said extraction reagent subsequent to
extraction of said antigen/analyte from said sample, and upstream
from a capture/detection reagent immobilised in said assay
device.
23. A method according to claim 22, wherein said method comprises
pretreating said substrate with a surface active agent.
24. A method for identifying carbohydrate antigens in a biological
sample, which method comprises applying said biological sample to a
sample receiving zone of a device according to any of claims 1 to
20 and monitoring for the presence of said antigen in said
detection zone.
Description
[0001] The present invention relates to a device and method for the
detection of carbohydrate antigens, and in particular antigens that
are characteristic of microbial/bacterial organisms, such as, for
example, the family Streptococcacae, including Group A and Group B
Streptococci.
[0002] Immunoassays for the detection of organisms, such as from
the family Streptococcae, typically rely on the detection of a
specific carbohydrate antigen. Antigenic macromolecules
(polysaccharides, polypeptides etc.) which append from the outer
surface of bacterial walls are specific to the bacterial species,
type group or strain. When these antigenic macromolecules are
exposed to the corresponding specific antibodies, the antibodies
and the antigens bind together to form a precipitin. However, the
antigenic molecules must first be released from the cell wall of
the organism before they can be detected. A number of methods exist
for carrying out such extraction treatments using for example,
nitrous acid, ProNase B enzyme, hot formamide or hot HCL. In the
case of nitrous acid, as the nitrous acid contacts the exterior
walls of the microorganism, the group antigen is released into
solution with its antibody specific reactivity intact.
[0003] The majority of immunoassay tests that utilise lateral flow
technology, for example, utilise nitrous acid for the extraction of
the carbohydrate antigen from the organism before contacting the
sample containing the extracted antigen with the assay device.
Nitrous acid, however, is chemically unstable. It is known only in
solution, yet quickly forms nitric acid and nitric oxide in the
presence of water. As a result, polysaccharides have conventionally
been extracted by the nitrous acid which is formed in a
pretreatment step by the reaction between an inorganic nitrate and
an aqueous acid, generated immediately prior to antigen extraction.
Following the extraction, the solution is then neutralised by the
addition of a neutralising agent, such as tris (hydroxymethyl)
aminomethane (TRIS) prior to the addition of the resultant solution
to the assay device.
[0004] Therefore, the extraction of carbohydrate antigen and its
subsequent detection would generally be as follows: a swab sample
is taken from the back of the throat, for example, and the swab
inserted into a vial containing nitrous acid generated by the
addition of aqueous solution of sodium nitrite and acetic acid. The
swab is subsequently agitated to release the antigens into
solution.
[0005] Following extraction of the antigen, the solution must be
neutralised prior to its addition to a lateral flow or other
suitable assay device. In the case of a lateral flow device, the
device will typically contain a mobilisable labelled binding
reagent capable of binding to the antigen as well as an immobilised
binding reagent capable of binding analyte provided downstream from
the labelled binding reagent. Thereafter, the antigen may be
detected in a conventional manner by formation of an immobilised
particulate labelled sandwich binding reagent complex. Such assay
devices are disclosed in EP291194.
[0006] The method described above, in addition to other prior art
methodologies, all require the separate steps of first extracting
the antigen by contacting the sample with the precursor nitrite and
acid. Both the acid and the nitrite may be contained in solution,
or alternatively one of the precursor reactants may be provided in
dried form and subsequently contacted with the remaining precursor
reactant contained in solution together with the sample.
Nevertheless, each of these methodologies require the subsequent
step of contacting the sample, following extraction of the antigen,
with the lateral flow assay device to detect the desired antigen
and which device is provided separately from the sample
extraction.
[0007] The present invention seeks to alleviate the problems
associated with such prior art methodologies and provides a kit and
assay device for the detection of antigens or analytes in an
organism, wherein the number of treatment steps are substantially
reduced and which kit and assay device are therefore particularly
easy to use. The test devices according to the invention find
particular utility for testing in a nurses or physicians office, a
remote or home setting and are therefore particularly useful and
suitable for use by non-specialist personnel.
[0008] Therefore, according to a first aspect, the invention
provides:
[0009] a lateral flow assay device for identifying carbohydrate
antigens in a biological sample, said device comprising a substrate
having
[0010] a) a sample receiving zone,
[0011] b) an extraction zone for receiving the sample from said
sample receiving zone and which extraction zone comprises
immobilised or otherwise absorbed therein at least one or more
reactants or reagents which when combined react to form an
extraction reagent for a desired antigen in said sample, [0012]
said sample to be added to said sample receiving zone optionally
comprising the remaining reactant required to form said extraction
reagent if not present in said extraction zone,
[0013] c) optionally, a neutralising agent capable of bringing the
pH of the resulting sample to within the operational pH range of
the assay, and
[0014] d) a detection zone for receiving a labelled specific
binding or capture reagent specific for said antigen to be
detected.
[0015] Lateral flow assay devices are known in the art and
generally comprise a porous material with a reagent containing
matrix incorporated therein. The sample is permitted to flow
laterally from the sample receiving zone downstream to a reaction
or detection zone comprising a capture reagent. In the operation of
such devices, a test sample is applied to a sample receiving area
or zone on the device and the sample moves or flows through the
porous material from the application or sample receiving zone to a
reaction or detection zone which includes therein an appropriate
capture reagent.
[0016] The extraction zone may be provided at or downstream from
the sample receiving zone.
[0017] In one embodiment of the present invention, the precursor
reactants are both separately provided in said extraction zone and
which when combined by virtue of the movement of the sample through
said substrate react to form said extraction reagent. Therefore,
when the solution of sample moves laterally through the assay
device it will contact each of the reactants in succession such
that when they combine they react to form the extraction reagent
which can act on the sample to extract any of the desired antigen
present. Therefore, the device of the present invention only
requires the step of adding the sample to the substrate thus
minimising the number of steps required and the amount of sample
and reactant manipulation.
[0018] The precursor reactants, or reagents, comprise an acid and
an acid generating reagent which acid generating agent is able to
react with the acid to form a nitrous acid.
[0019] The acid is preferably immobilised in the extraction zone of
the device by drying, or the like, and is provided separate from
the other reagent, such that when the sample moves through the
lateral flow assay device, it will come into contact with each of
the reagents in succession, both of which will then combine to form
the nitrous acid which can then function to extract the antigen
from the organism to permit detection.
[0020] The immobilised acid may be any suitable acid which is
capable of reacting with an acid generating reagent to produce
nitrous acid, such as a polycarboxylic acid, a polysulfonic acid, a
polystyrenesulphoic acid, a polyphosphoric acid, a polyacrylic
acid, and a polymethacrylic acid. Other such acids are known to
those of skill in the art.
[0021] The acid however, preferably comprises a non-volatile
organic, acid such as any of citric acid, malonic acid,
phenylacetic acid, oxalic acid, glycolic acid, chloroacetic acid,
trichcroacetic acid, fluoroacetic acid, bomoacetic acid, iodaacetic
acid, succinic acid, glutaric acid, adipic acid, pimelic acid,
suberic acid, benzoic acid, benzene sulfonic acid, p-toluene
sulfonic acid, azelaic acid and sebacic acid. However, a polymeric
acid, such as polysulphonic acid is particularly preferred and may
be provided on said assay device in the form of a resin. An example
of a suitable resin is polystyrene.
[0022] The acid may be a polymeric acid and may be immobilised by
conjugation to particles. Where the acid is conjugated to a
particle, the particle size may, advantageously, be chosen so that
it is larger than the pore size of the lateral flow assay such that
it is unable to move along the length of the porous carrier with
the fluid sample.
[0023] Alternatively, the acid may form part of the sample fluid
carrying matrix such as a sample receiving wick, or the porous
carrier itself. This may be achieved, for example, by sulphonation
of the material comprising the particular porous matrix.
[0024] Any suitable acid generating reagent which is stable in the
dry state and capable of reacting with the immobilised acid to
produce nitrous acid may be used, such as an alkali metal
nitrite.
[0025] The acid generating reagent, preferably, comprises a
nitrite, which may include any of, inorganic nitrites such as
sodium, potassium, lithium, calcium, strontium, barium and silver
nitrites, as well as organic nitrites such as butyl and isoamyl
nitrites. Sodium and potassium nitrites are preferred while sodium
nitrite is most preferred. The concentration of nitrite and organic
acid combined in the extraction reagent can be varied widely
depending on the particular compounds used, their water solubility
and the suspected amount of antigen present in the test sample.
[0026] The sodium nitrite may be added to the device in either an
aqueous or dry state.
[0027] A neutralising agent may also be provided where necessary to
bring the fluid sample after it has contacted the extraction zone
to within the operational pH range of the assay. The pH range of
the assay may vary and will be dependent upon the nature and type
of the capture reagents (for example the particular antibody type
and whether it is polyclonal or monoclonal) as well as the antigen
to be determined. In some circumstances a neutralising agent may
not be required should the pH of the resultant sample mixture fall
within the operational pH range of the assay.
[0028] However, a neutralising agent is preferred as it allows the
pH of the reaction mixture to be optimised which in turn allows for
optimisation of the assay sensitivity. A typical pH range may be
between pH 6-9. The neutralising agent where present is preferably
buffered. Many such neutralising buffers are known in the art,
although a preferred one is tris (hydroxymethyl) aminomethane
(TRIS).
[0029] The location of the neutralising agent, where present, will
be determined by the operational pH range of the capture reagents.
Thus, the neutralising agent may be provided downstream from the
extraction zone and upstream from a capture reagent. Alternatively,
the neutralising agent may be provided downstream from a first
capture reagent and upstream from a second capture reagent.
Preferably it is provided within the device in the dry state prior
to use.
[0030] In one embodiment of the invention, the detection zone may
comprise a labelled binding reagent specific for said antigen to be
detected, and which binding reagent may be provided in dried or
otherwise immobilised form within the detection zone.
Alternatively, the labelled specific binding reagent may be added
subsequently to the detection zone prior to addition of the sample
to be tested. Therefore, the device will, typically, comprise a
capture reagent that may, preferably, be first immobilised in the
detection zone and which is capable of capturing a particular
analyte or antigen of interest. The device may alternatively
comprise a mobilisable labelled capture reagent located at a
position at or upstream from the detection zone and which is also
capable of capturing the analyte or antigen of interest.
[0031] In the situation wherein a mobilisable labelled capture
reagent is provided in the device prior to use, this reagent is
preferably located at a position downstream from the optional
neutralising reagent. The mobilisable capture reagent may be
provided on and/or in a separate porous carrier arranged so as to
be in fluidic contact with and upstream from the porous carrier,
such as disclosed by U.S. Pat. No. 6,352,862. The separate porous
carrier may be macroporous and may be of a different material to
that of the porous carrier. According to one embodiment, the porous
carrier is nitrocellulose and the macroporous carrier is chosen
from one of a glass fibre or a synthetic polymeric material, such
as polypropylene.
[0032] Alternatively, the mobilisable capture reagent may be
provided in the region of the sample receiving wick or the porous
carrier.
[0033] The capture reagent may be chosen from any species that is
capable of forming a binding pair with the carbohydrate antigen and
may be, for example, an antibody or fragment thereof, a lectin,
boronic acid or boronic acid derivative. The capture reagent may be
a specific capture reagent. The antibody may be either polyclonal
or monoclonal.
[0034] The presence or amount of analyte in the sample may be
determined by the detection of the labelled capture reagent present
in the detection zone. The labelled capture reagent may be
determined visually or by other suitable means.
[0035] The label may be chosen from any suitable label, such as
dyed latex spheres or beads or dye imbibed liposomes. The
particular label may also comprise a metal sol, such as a gold
sol.
[0036] A control zone may also be provided in the device according
to the invention, which control zone may be provided at a position
at or downstream from the detection zone. The control zone serves
to indicate whether the test has been carried out correctly.
Typically, the control zone comprises a further immobilised capture
reagent which is able to capture the labelled capture reagent.
[0037] In one embodiment of the device of the present invention,
the porous material, preferably comprises a porous wick, which can
be made or obtained from any bibulous, porous or fibrous material
capable of absorbing liquid rapidly. The porosity of the material
can be unidirectional (i.e. with pores or fibres running wholly or
predominantly parallel to an axis of the member) or
multidirectional (omni directional, so that the member has an
amorphous sponge-like structure). Porous plastics material, such as
polypropylene, polyethylene (preferably of very high molecular
weight), polyvinylidene fluoride, ethylene, vinyl acetate,
acrylonitrile and polytetrafluoro-ethylene can be used.
[0038] In an alternative embodiment, however, the assay device may
comprise one or more microfluidic channels, the dimensions of which
are such as to permit a liquid sample to move through the one or
more channels by capillary action. The one or more capillary
channels may be provided in addition to or instead of the one or
more porous or absorbent matrices forming part of the device.
[0039] It can be advantageous to pre-treat the carrier material
with a surface active agent during manufacture, as this can reduce
any inherent hydrophobicity in the carrier material and therefore
enhance its ability to take up and deliver a moist sample rapidly
and efficiently. Porous sample receiving members can also be made
from paper or other cellulosic materials, such as
nitro-cellulose.
[0040] If desired, an absorbent "sink" can be provided at the
distal end of the carrier material. The absorbent sink may comprise
of, for example, Whatman 3MM.RTM. chromatography paper, and should
provide sufficient absorptive capacity to allow any unbound
labelled capture reagent to wash out of the detection zone.
[0041] As an alternative to such a sink it may be sufficient to
provide a length of porous solid phase material which extends
beyond the detection zone.
[0042] According to a second aspect of the invention, there is
provided a kit for identifying carbohydrate antigens in a
biological sample, which kit comprises a device as herein before
described and means for contacting said sample with said sample
receiving area.
[0043] Also provided by a further aspect of the invention is a
method of producing an assay device according to the invention,
which method comprises immobilising in a lateral flow assay device
having a sample receiving area, one or more reagents in an
extraction zone of said device and which reagents when combined
form an extraction reagent for a specific carbohydrate antigen in a
sample added to said sample receiving zone, providing a
neutralisation buffer downstream of said extraction zone to
neutralise the pH of said extraction reagent subsequent to
extraction of said antigen from said sample, and upstream from a
capture/detection reagent immobilised in said assay device.
[0044] In an even further aspect, the invention comprises a method
for identifying carbohydrate antigens in a biological sample, which
method comprises applying said biological sample to a sample
receiving zone of a device as herein before described, and
monitoring for the presence of said antigen in said detection
zone.
[0045] The present invention may be more clearly understood from
the following exemplary embodiment which is provided by way of
example only, with reference to the accompanying drawings
wherein;
[0046] FIG. 1. is an illustration of a device according to the
invention.
[0047] FIG. 2 is an illustration of the visual results obtained
using an assay device of the invention to detect Group A and C
Streptococci.
[0048] In the device according to the invention as illustrated in
FIGS. 1 to 2, there is provided a lateral flow assay device
generally indicated by the reference numeral 1, comprising a sample
receiving zone or area 2 and an extraction zone 4 for receiving
sample moving laterally through the device 1 in the direction from
the sample receiving zone to a detection zone 8. In the extraction
zone there is provided in a dried form or otherwise immobilised
therein an acid generating reagent 4a and an organic acid 4b. A
preferred acid generating reagent is sodium nitrite whereas the
preferred acid is a polysulfonic acid provided in the form a resin.
The polysulfonic acid is provided in a groove or channel 5 cut or
etched in the device.
[0049] A neutralising buffer 6 is provided downstream of the acid
generating reagent and upstream (as defined by the direction of
flow in the assay device) of a capture reagent provided in the
capture or detection zone 8.
[0050] The capture or detection reagent comprises a labelled
antibody specific for the antigen to be detected and which can be
visualised by virtue of an antigen-antibody complex in the
detection/capture zone. Preferably, the antibody is specific for
Streptoccal A antigen.
Experimental
[0051] A groove of approx 2 mm wide.times.3 mm deep was made at a
distance of 10 mm from the near end of a porous sample receiving
wick of dimensions 48 mm in length.times.7 mm width.times.4 mm
depth (Filtrona, Richmond Inc, Colonial Heights, Va., USA).
[0052] 250.mu. 1M sodium nitrite was applied to one end of the wick
and allowed to soak in. 200 .mu.l of 1.6M TRIS pH 8.5 was
subsequently applied to the wick at the top end and allowed to soak
in, as indicated in FIG. 1. The wick was dried at 50.degree. C.
overnight. Approximately 20 mg of sulfonic acid resin (MP 70-90
mesh Novabiochem cat. No. 01-64-0432 1.6 mmole/g) was subsequently
packed into the groove.
Preparation of A Porous Release Matrix Comprising A Mobilisable
Particulate Labelled Binding Reagent
[0053] Anti Strep A carbohydrate antibody (Biotech Inc.) was
absorbed onto 400 mm blue latex particles (Duke Scientific, USA) at
a concentration of 30 .mu.g/ml (0.5% latex solids) was prepared
containing the freeze dried latex particles coated with the
antibody. The antibody sensitised latex particles were infused into
a glass fibre pad of dimensions 20 mm.times.8 mm.times.1.2 mm pad
having an open structure (Sintair, USA) and freeze dried.
[0054] The wick containing the dried extraction reagents was
subsequently placed on top of the prepared glass fibre pad.
Preparation of the Porous Carrier Comprising An Immobilised Capture
Reagent
[0055] Anti Strep A polyclonal antibody was deposited by means of a
microprocessor-controlled microsyringe onto an 8 mm wide porous
nitrocellulose carrier strip (Schleicher and Schuell based 8.mu.
porosity) at a concentration of 1.9 mg/ml and at a flow rate of 0.8
.mu.l/mm.
[0056] The antibody was applied in the form of a stripe oriented
perpendicular to the flow of liquid sample at a detection zone
located downstream from the labelled antibody and the antibody was
subsequently blocked with 1% (w/v) polyvinyl alcohol/3% (w/v)
sucrose to immobilise it to the porous carrier.
Construction of the Device
[0057] Lateral flow carrier devices were constructed as shown in
FIG. 1 using the materials as prepared above. A porous sample
receiving wick containing the dried extraction reagents was placed
on top of a glass pad containing the freeze dried antibody
sensitised latex particles coated with anti Strep A polyclonal
antibody.
[0058] The glass pad was subsequently placed in contact with the
nitrocellulose porous carrier such that liquid sample applied to
one end of the porous sample receiving wick would be capable of
permeating into the glass fibre pad and along the nitrocellulose
porous carrier.
[0059] A further anti mouse antibody was immobilised at a location
downstream from the immobilised capture reagent which functioned as
a control zone. Finally an absorbent sink pad was placed in contact
with the distal end of the nitrocellulose strip.
[0060] A further device was also constructed for the detection of
Streptococcus C as described above except the anti Strep A
antibodies were replaced by anti-Strep C antibodies.
[0061] Samples of Strep A and C were respectively diluted to 10e9
cfu/ml in water. The bacterial suspensions (100 .mu.l) were applied
to the near end of the device (sodium nitrite end), followed by
1000 .mu.l of water (to move the liquid through the device).
[0062] As a control the test was repeated except that 100 .mu.l of
water containing no bacteria was applied to a device. The results
of the tests are shown by FIG. 2. As may be seen from FIG. 2, the
presence of blue latex sensitised antibodies may be observed at the
detection zone indicating the presence of Strep A and C
carbohydrate antigen.
[0063] Thus results show that the respective carbohydrate antigens
were released from the bacteria by nitrous acid generated by
re-hydration of the reagents contained within the device, and that
these antigens were then able to be specifically detected in the
subsequent immunoassay.
* * * * *